Ex-vivo priming for generating cytotoxic t lymphocytes specific for non-tumor antigens to treat autoimmune and allergic disease

ABSTRACT

Cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derived from IgE molecule can be generated in vitro by stimulating resting naive CD8 T cells with IgE peptides presented by artificial antigen presenting cells. The IgE specific CTLs lyse the target cells loaded with IgE peptides in vitro and inhibit antigen specific IgE response in vivo. In addition, adoptive transfer of the IgE specific CTL to an asthmatic mouse model can inhibit the development of lung inflammation and airway hypersensitivity. IgE specific CTL provides a treatment for allergic asthma and other IgE-mediated allergic diseases. Antigenic peptides identified from non-tumor self-antigens induce specific cytotoxic T lymphocyte (CTL) in vitro. The CTL induced by peptides identified from CD40L can kill activated CD4 T cells. In vitro generated CTL specific for CD40L inhibit CD4-dependent antibody responses of all isotypes in vivo. In contrast, CTL induced by antigenic peptides derived from IgE specifically inhibit IgE responses, and adoptive transfer of CD40L-specific CTL to NOD mice at early age delay the development of diabetes in NOD mice. In vitro generated CTL specific for non-tumor self-antigens expressed on activated CD4 T cells regulate immune responses in vivo.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. provisional application Ser.No. 60/291,300, filed May 15, 2001, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Immune responses to foreign antigens such as those found in bacteria andvirus protect from and eliminate infections. However, aberrant immuneresponses can cause allergic diseases and autoimmune diseases. Immuneresponses to foreign, sometimes innocuous, substances such as pollen,dust mites, food antigens and bee sting can result in allergic diseasessuch as hay fever, asthma and systemic anaphylaxis. Immune responses toself-antigens such as pancreatic islet antigens and cartilage antigenscan lead to diabetes and arthritis, respectively. The hallmark of theallergic diseases is activation of CD4 T cells and high production ofIgE by B cells, whereas the salient feature of autoimmune diseases areactivation of CD4 T cells and over production of inflammation cytokines.The current therapies have been focused on the treatment of symptoms ofallergy and autoimmune diseases and do not prevent the development andprogression of the diseases.

CTLs are derived from resting naïve CD8 T cells and recognize antigenicpeptides presented by Major Histocompatibility Complex (MHC) class Imolecules. When resting CD8 T cells encounter antigenic peptides/MHCcomplex presented by professional antigen presenting cells, CD8 T cellswill be activated and differentiated into armed CTL. Upon recognition ofpeptide/MHC complexes on the target cells, the antigen specific CTL willdeliver a lethal hit and lysis the antigen-expressing target cells, suchas virus infected target cells or tumor cells. Activation of naive Tcells in vivo is controlled by multiple receptor-ligand interactionsbetween T cells and professional APC such as dendritic cells (R. M.Steinman, Annu. Rev. Immunol. (1991) 9:271-296). It is generallyaccepted that two signals are required for activation of naive T cells(C. A. Janeway and K. Bottomly, Cell (1994) 76:275-285). Signal 1 isinduced by the interaction between TCR and MHC/peptide complexes (R. N.Germain, Cell (1994) 76:287-299) and is aided by binding of CD4/CD8co-receptors to non-polymorphic regions of MHC class II/I molecules,respectively (M. C. Miceli and J. R. Parnes, Adv. Immunol. (1993)53:59-122). Signal 2 is qualitatively different from Signal 1 and isdelivered via T cell costimulatory molecules interacting withcomplementary ligands on APC, e.g. through CD28 interaction with B7 (P.S. Linsley and J. A. Ledbetter, Annu. Rev. Immunol. (1993) 11:191-212;Lenschow et al., Annu. Rev. Immunol. (1996) 14:233-258). Signals 1 and 2function synergistically and trigger a series of signaling events whichultimately induce T cells to proliferate, produce cytokines anddifferentiate into effector cells (Mueller et al., Annu. Rev. Immunol.(1989) 7:445-480; A. Weiss and D. R. Littman, Cell (1994) 76:263-274).The relationship between Signals 1 and 2, however, is unclear.

Although a variety of molecules have been reported to have costimulatoryfunction, particular attention has been focused on costimulationdelivered via CD28-B7 interaction (R. H. Schwartz, Cell (1992)71:1065-1068). CD28 is a molecule with a single Ig like domain and isconstitutively expressed as a homodimer on T cells (P. S. Linsley and J.A. Ledbetter, (1993) supra). Through its interaction with either B7-1 orB7-2 molecules on APCs, CD28 molecules are thought to transduce uniquesignals that stimulate T cell to produce growth-promoting cytokines suchas IL-2 (June et al., Immunol. Today (1994) 15:321-331), to upregulateexpression of survival factors such as Bcl-X_(L) (Boise et al., Immunity(1995) 3:87-98) and to prevent anergy induced by Signal 1 alone (R. H.Schwartz, Curr. Opin. Immunol. (1997) 9:351-357).

Another pair of molecules that has an important role in T cellactivation is LFA-1/ICAM-1 (Van Seventer et al., J. Immunol, (1990)144:4579-4586). ICAM-1 belongs to the Ig gene superfamily and has fiveIg C like domains in the extracellular regions; it is expressed on bothhemapoietic and nonhemapoietic cells. The receptor for ICAM-1 on T cellsis LFA-1 (CD11/CD18), which belongs to the b2 integrin family (T. A.Springer, Cell (1994) 76:301-314). The interaction of LFA-1 with ICAM-1has potent costimulatory function on T cells (Shimizu et al., Immunol.Rev. (1990) 114:109-143), although opinions vary on whether thisfunction reflects a separate signaling pathways or increased adhesionbetween T cells and APC (Damle et al., J. Immunol. (1993) 151:2368-2379;Bachmann et al., Immunity (1997) 7:549-557).

In addition to B7 and ICAM-1 molecules, several other molecules on APCs,including CD70 (Hintzen et al., J. Immunol. (1995) 154:2612-2623) andheat-stable antigen (HSA) (Liu et al., J. Exp. Med. (1992) 175:437-445),can exert quite potent costimulatory function through their interactionwith their respective ligands on T cells. The implication is that T-APCinteraction is highly complex and involves multiple interactions betweencomplementary sets of molecules on T cells and APCs. The interaction ofeach set of molecules could trigger specific signals which inducedifferent cellular events. The combination of the different signals mayact synergistically for optimal T cell activation and determine thefinal fate of T cells. Alternatively, the function of costimulationmolecules may be redundant and the signals induced by each set ofcostimulation molecules are additive. The requirement for each set ofcostimulation molecules will be influenced by the strength andcharacteristics of Signal 1.

In considering these two possibilities, it is important to understandthe minimal requirements for stimulating naive T cells. Studies withCD28^(−/−) mice indicated that CD28-B7 interaction is highly importantin some situations, but not in others (Shahinian et al., Science (1993)261:609-612). Likewise, the requirement for LFA-1/ICAM interaction inprimary responses is not an invariable finding (Shier et al., J.Immunol. (1996) 157:5375-5386).

CD8 T cells recognize antigenic peptides derived mainly from virusproteins and proteins expressed on tumor cells. However, it has recentlybeen reported that newly synthesized proteins are preferentiallyprocessed by antigen-processing machinery (Schubert et al., Nature,(2000) 404:770-774). Upon activation, immune cells have acquired theability to synthesize a number of new proteins, it is possible that IgEproducing B cells and activated CD4 T cells would present a differentsets of peptide/MHC complexes than the non-IgE producing cells andresting CD4 T cells. These peptides/MHC complexes presented on IgEproducing B cells and activated CD4 T cells would be able to berecognized by CD8 T cells. Thus, CTL specific for these peptides/MHCcomplexes would be able to treat allergy and autoimmune diseases.However, a number of tolerance mechanisms have been able to prevent theactivation the CD8 T cells towards self-antigens in vivo.

CD8 lymphocytes (CTLs) are the arm of adaptive immunity responsible forthe recognition and elimination of infected cells, tumor cells, andallogeneic cells. Once primed, CTL can recognize their target antigen ona wide variety of cells and accomplish their function by lysing thetarget cell and/or secreting cytokines like TNF-alpha, or IFN-gamma.

Presentation of antigen to CD8⁺ CTL (cytotoxic T lymphocytes) occurs inthe context of MHC class I molecules (MHC-I), while presentation ofantigen to CD4⁺ HTL (helper T lymphocytes) occurs in the context of MHCclass II molecules.

Efficient induction of CD4⁺ T cell requires that the T cells interactwith antigen presenting cells (APC) i.e. cells that express MHC class IIand co-stimulatory molecules. APC are dendritic cells, macrophages andactivated B cells. Although nearly all nucleated cells express MHC-I,naive CTL also require presentation of antigen (Ag) by bonemarrow-derived APC for efficient priming (Dalyot-Herman et al., J.Immunol., 165 (12):6731-6737). Dendritic cells are highly potentinducers of CTL responses (J. Bancherean and R. M. Steinman, Nature,(1998) 392:245-252) and are thought to be the principal APC involved inpriming CTL. Once primed, CTL can recognize their cognate Ags on a widevariety of cells and respond by lysing the target cell and/or secretingcytokines.

Although bone marrow-derived APC are required to efficiently prime CTLresponses (P. J. Fink and M. J. Bevan, Exp. Med. (1978) 148:755-766)activated CTL are readily able to recognize and respond to Ag presentedby a wide variety of cells. Induction of tumor- or viral-specific CTLimmune responses in vivo have been shown to be dependent on bone marrowderived antigen-presenting cells (Paglia et al., J. Exp. Med. (1996) 183(1):317-322; Labeur et al., J. Immunol. (1999) 162 (1):168-175). It isgenerally accepted that bone marrow derived APC, through mechanismsunique to these cells, take up cellular antigens either in the form ofsoluble antigen, associated with chaperone molecules or by phagocytosis.

It has long been demonstrated that responses to cellular antigens aredependent on help delivered by CD4⁺ T cells. It has also been shown thatthe cellular antigen had to be presented on the same APC for recognitionby the CTL and the HTL. The nature of this help has been interpreted asa need of IL-2 necessary for CTL expansion. Recent studies have shownthat this help results from the activation of dendritic cells by HTL andis mediated via CD40-CD40L interaction (S. R. Clarke, J. Leukocyte Bio.(2000) 67 (5):607-614).

A likely scenario for the induction of a CD8 mediated immune response toa cellular antigen (derived from a tumor cell or an infected cell) istherefore the following: dendritic cells acquire antigens derived fromtumor or infected cells. Interaction of DC-antigen with CD4 cells enablethe DC to activate the CD8 cells.

SUMMARY OF THE INVENTION

Immune cells, such as IgE producing B cells and activated CD4 T cellsplay a central role in the pathogenesis of allergic diseases andautoimmune diseases. The present invention utilizes cytotoxic Tlymphocytes (CTLs) to eliminate or inhibit the immune cells that causethe allergy and/or autoimmune diseases. Thus, the development andprogression of diseases can be prevented or interrupted by the methodsof the present invention.

The present invention provides a method for producing CTL specific forone or more non-tumor self antigen T cell epitopes, comprising:

-   -   a. isolating CD8⁺ T cells from a subject;    -   b. loading antigen presenting cells (APC's) having Class I MHC        molecules with the T cell epitopes;    -   c. culturing the CD8⁺ T cells with the antigen-loaded APC's for        a period of time sufficient for activation of precursor CD8⁺ T        cells specific for the T cell epitopes;    -   d. expanding in culture the activated CD8⁺ T cells in the        presence of components required for proliferation of the        activated CD8⁺ T cells; and,    -   e. collecting CD8⁺ T cells from the culture.

The present invention also provides CD8⁺ T cells that are specificallycytotoxic for a disease causing target cell, wherein the target cell hason its surface one or more non-tumor self antigen T cell epitopesassociated with Class I MHC molecules, and wherein the CD8⁺ T cells havebeen selectively activated by interaction with Class I MHC moleculesassociated with the non-tumor self antigen T cell epitopes.

The present invention also provides a method for treating a diseasemediated by a disease causing target cell, wherein the target cell hason its surface one or more non-tumor self antigen T cell epitopesassociated with Class I MHC molecules, comprising administering to apatient in need of such treatment, activated CD8⁺ T cells wherein theCD8⁺ T cells have been selectively activated by interaction with Class IMHC molecules associated with the non-tumor self antigen T cellepitopes.

The present invention demonstrates that by making and using artificialantigen presenting cells, tolerance of CD8 T cells to self antigens wasbroken and CTLs specific for antigenic peptides identified from IgE orCD40L proteins were generated. Adoptive transfer of the in vitrogenerated CTLs specific for CD40L to NOD mice dramatically delayed thedevelopment of diabetes, and CTLs specific for IgE peptides inhibitedthe production of IgE and reduced lung inflammation in an asthmaticmouse model. The above system is potentially applicable to humandiseases that are caused by CD4 T cells and by IgE producing B cells.Autoimmune diseases that caused by CD4 T cells are diabetes, rheumatoidarthritis, SLE, multiple sclerosis and psoriasis. Whereas allergicdiseases mediated by IgE are systemic anaphylaxis caused by drugs,venoms and peanuts, allergic rhinitis, food allergy, and allergicasthma. In addition other self-antigens that expressed on immune cellscan also be used for generation of CTLs in vitro as well in vivo fortreatment of autoimmune diseases and allergic diseases. Antigenicpeptides, proteins or RNA and DNA encoding the non tumor antigensexpressed in non tumor cells can also be used to develop vaccines fortreatment or prevention of allergy and autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The amino acid sequences of IgE^(a) SEQ ID NO: 14 and IgE^(b)SEQ ID NO: 50 constant regions were aligned with vector NTI software.The sequence differences between the two alleles are bold andunderlined.

FIG. 2, Panels A, B, C and D:

-   -   CD8⁺ T cells were purified from lymph nodes of CBF1/J mice (A, B        and D) or from B6, interferon γ knock out mice (IFNγ^(−/−)) or        perforin (PF^(−/−)) knock out mice (C). The purified CD8 T cells        were cultured with indicated IgE peptides presented by SC2 cells        transfected with D^(b) MHC class I, B7-1 (CD80) and ICAM-1        (CD54) molecules. Low dose of recombinant IL-2 (20 units/ml) was        added to the culture at Day 3 and every other day thereafter. On        Day 9, CTL activity was measured against ⁵¹Cr labeled RMAS cells        loaded with or without indicated IgE peptides. In FIG. 2, Panel        D, anti-D^(b) mAb (20 μg/ml) was added at the beginning of CTL        assay.

FIG. 3: Adult CBF1/J mice (8 to 12 weeks) were immunizedintraperitoneally with 50 μg ovalbumin (OVA) precipitated with AlumHydroxide on Day 1 and Day 14 respectively. Serum IgE, IgG1 and IgG2awere measured by ELISA on Day 28. Two weeks after the secondimmunization, the mice were challenged with OVA intranasally every otherday for three treatments. IgE-specific CTLs or control CTLs (5×10⁶) weregive intravenously one day after each challenge. Serum IgG and IgE weremeasured again two weeks after the last CTL therapy.

FIG. 4, Panels A, B, C and D:

-   -   CBF1/J mice were immunized as in FIG. 3. Two weeks after the        second immunization, two different doses (5×10⁶ and 10×10⁶) of        anti-IgE CTLs were given intravenously three times every other        day. Three weeks after the CTL treatment, serum IgE and        OVA-specific IgE was measured and challenged with OVA        intranasally every other day for three treatments. After the        last challenge, bronchial alveolar lavage (BAL) was collected        and the total cells in BAL were counted. Eotaxin in the BAL was        measured by ELISA and Eosinophils cells in the BAL were        differentiated by HE staining.

FIG. 5, Panels A and B:

-   -   CBF1/J mice were immunized with OVA/Alum at Day 1 and Day 14.        Two weeks after the second immunization, mice were injected        every other day for three treatments with PBS, anti-IgE CTL or a        control CTL (anti-influenza CTL) as indicated. Three weeks after        the last treatment, mice were challenged with OVA intranasally        every other day for three treatments. One day after the last        challenge with OVA, airway responsiveness to methacholine for        each mouse was measured by whole body plethrography. Two        independent experiments were shown in Panels A and B        respectively.

FIG. 6, Panels A and B:

-   -   Adult CBF1/J mice (8 to 12 weeks) were immunized        intraperitoneally with 50 μg ovalbumin (OVA) precipitated with        Alum Hydroxide on Day 1 and Day 14 respectively. Two weeks after        the second immunization, the mice were given IgE-specific CTLs        (5×10⁶) or PBS intravenously. Three weeks after the last        treatment, mice were challenged with OVA intranasally every        other day for two to three treatments. One day after the last        challenge with OVA, the BAL was prepared from each mouse and the        lung from each mouse was fixed and stained with HE. A        representative HE staining of lung tissue from mice received PBS        (Panel A) or from mice received anti-IgE CTL (Panel B) was        shown.

FIG. 7: The amino acid sequence deduced from cDNA encoding the human IgEconstant region. Total RNA was prepared from U266 cell line, whichproduces human IgE. The total RNA was reverse transcribed and amplifiedby PCR with two oligoes encoding the 5′ and 3′ human IgE constant regionrespectively. The cDNA was cloned into pcDNA3 vector and sequenced.

FIG. 8: Drosophila cells transfected with human HLA-A2 class I cDNA werecultured with a titrated concentration of indicated IgE peptides orcontrol peptide (H690) overnight at room temperature and furthercultured at 37° C. for an additional two hours. The cells were washedand stained with anti-HLA-A2 mAb and analyzed by flow cytometry. Themean fluorescence intensity was indicated at Y axis and the peptideconcentration was indicated at X axis.

FIG. 9, Panels A, B, C and D:

-   -   CD8 T cells were purified from individual donors and cultured        with Drosophila cells transfected with HLA-A2, hB7-1, hB7-2,        hICAM-1 and hLFA-3 molecules in the presence of indicated        peptides. After being cultured for six days, low doses of hIL-2        was added to the culture and re-stimulated with peptides loaded        autologous adherent cells for an additional seven days. The CTLs        were then harvested and the specific killing activities were        tested with ⁵¹Cr labeled T2 cells that loaded with indicated        peptides by a standard chromium release assay.

FIG. 10: The amino acid sequence of human IgE was derived as describedas in FIG. 6. The antigenic peptides that contain nine amino acids wereunderlines and the antigenic peptides that contain ten amino acids wereshown in bold.

FIG. 11: TAP 2 deficient RMA.S cells (right panel) or L^(d) transfectedRMA.S cells (left panel) were incubated with indicated concentration ofpeptides at 28° C. overnight and then incubated at 37° C. for two tofour hours. The cells were harvested and stained with mAb specific forL^(d) (right panel) or for D^(b) (left panel) and analyzed with FACScan.

FIG. 12: CD8⁺ T cells were purified from LN of B10.D2 mice and culturedwith Drosophila cells transfected with L^(d), B7-1 and ICAM-1 in thepresence of CD40L.186 peptide (left panel) or QL9 peptide (right panel).IL-2 (20 U/ml) was added to the culture at Days 3 and 5. On Day 7, CTLactivity was measured against ⁵¹Cr labeled RMAS.L^(d) target cells inthe presence of indicated peptides.

FIG. 13: Purified CD8⁺ T cells from B6 mice were cultured withDrosophila cells transfected with D^(b), B7-1 and ICAM-1 in the presenceof Ig E.44 peptide (left panel) or Ig E.366 peptide (right panel). IL-2(20 U/ml) was added to the culture on Days 3 and 5. CTL was harvested onDay 7 and their specific activity was measured against ⁵¹Cr labeledRMA.S target cells in the presence of indicated peptides.

FIG. 14, Panels A and B:

-   -   Purified CD4 or CD8 T cells were activated with plate-bound        anti-CD3 and anti-CD28 for forty hours (top panel) or for        indicated time (bottom panel) and were stained with indicated        mAb.1

FIG. 15: CD40L specific CTL were generated as described in FIG. 2. CD4cells used as targets were purified from wild type, CD40L^(−/−) orμ2m^(−/−) mice and activated with anti-CD3 and anti-CD28 for fortyhours.

FIG. 16: B10.D2 (top panel) or B6 (bottom panel) were immunized withOVA+CFA and treated with Ab or CTL as indicated. The spleen cells weremeasured for OVA-producing B cells by ELISA spot at Day 21 afterimmunization.

FIG. 17, Panels A, B, C, D and E

-   -   B10.D2 mice were immunized with OVA+CFA on Day 1. Anti-CD40L CTL        or anti-CD40L Ab were given at Days 1, 3, 5. Serum was collected        on Day 14 and OVA-specific immunoglobulins were measured by        ELISA.

FIG. 18, Panels A, B and C:

-   -   CD8 T cells were purified from C57BL/6 mice and cultured with        Drosophila cells transfected with D^(b), B7-1 and ICAM-1 in the        presence of IgE.44 peptide (A), IgE.366 peptide (B) and IgE.125        (C). IL-2 (20 units/ml) was added to the culture on Day 3 and 5.        CTLs were harvested on Day 7 and their specific killing activity        was measured against ⁵¹Cr labeled RMA.S target cells in the        presence or absence of indicated peptides.

FIG. 19: CD8 T cells were purified from C57BL/6 (B6), perforin knock outmice (pf^(−/−)) and IFNγ knock out mice (IFNγ^(−/−)) were cultured withDrosophila cells transfected with Db, B7-1 and ICAM-1 in the presence ofIgE.44 peptide. IL-2 (20 units/ml) was added to the culture on Day 3 and5. CTLs were tasted on Day 7 and their specific killing activity wasmeasured against 51Cr labeled RMA.S target cells in the presence orabsence of IgE.44 peptide. In Panel A, CTL activity was measured in thepresence or absence of 10 μg/ml of anti-D^(b) monoclonal antibody.

FIG. 20: CD19⁺ B cells were purified from human PBMC and cultured withIL-4 (100 ng/ml) and anti-CD40 mAb (5 mg/ml). Anti-IgE CTLs weregenerated as described on FIG. 9 in the presence of indicated IgEpeptides (B) IgE47 and 96, (C) IgE 884 and 890. CTLs were added at Day 4to the culture B and C. On Day 6, the culture supernatant was collectedand human IgE was measured by ELISA. In culture A, no CTLs were addedand no B cells in culture D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides in one embodiment, a method for treatinga subject with non-tumor self-antigen T cell epitopes comprising:

-   -   a. preparing a naturally occurring antigen presenting cell (APC)        or a non-naturally occurring antigen-presenting cell line        (nnAPC), wherein said APC or said nnAPC is capable of presenting        up to about fifteen different peptide molecules that is        associated with allergic and/or autoimmune disease, preferably        about ten different peptide-epitope molecules, simultaneously        where each peptide is about six to twelve amino acids in length,        preferably about eight to ten amino acids in length and in a        concentration range of about 10 nM to 100 μM;    -   b. harvesting CD8⁺ cells from said subject or a suitable donor;    -   c. stimulating said CD8⁺ cells with said APC or said nnAPC cell        line;    -   d. adding said CD8⁺ cells to media that contains a cytokine,        such as, IL-2, IL-7 or CGM, preferably, IL-2, or IL-2 and IL-7        in combination;    -   e. mixing unsuspended peripheral blood monocytes, or        alternatively, CD8-depleted peripheral blood monocytes collected        from said subject or a suitable donor with about 10 to 50 μg/ml        of a peptide;    -   f. irradiating said peripheral blood monocyte suspension with a        sufficient dose of γ-radiation necessary to sterilize all        components in the suspension, except the desired peripheral        blood monocytes, such as a dose in the range of about 3,000 to        7,000 rads, preferably about 5,000 rads;    -   g. isolating adherent peripheral blood monocytes;    -   h. loading said adherent peripheral blood monocytes with about        10 ng/ml to 10 μg/ml of said each peptide;    -   i. combining said CD8⁺ cells with said adherent peripheral blood        monocytes at a ratio of about ten CD8⁺ cells to one peripheral        blood monocyte;    -   j. optionally stimulating said combined suspension of CD8⁺ cells        and peripheral blood monocytes for about six to seven days;    -   k. optionally stimulating said suspension of CD8⁺ cells and        peripheral blood monocytes with IL-2 and IL-7 in media;    -   l. optionally assaying CD8⁺ suspension for suitable CTL        activity, and optionally assaying for CTL purity, sterility and        endotoxin content; and    -   m. inoculating said subject with CD8⁺ suspension.

Another embodiment of the present invention provides a method fortreating a subject comprising,

-   -   a. preparing a naturally occurring antigen presenting cell (APC)        or a non-naturally occurring antigen-presenting cell line        (nnAPC), wherein said APC or said nnAPC is capable of presenting        up to about fifteen different peptide-epitope molecules that is        associated with allergic and/or autoimmune disease, preferably        about ten peptides, simultaneously where each peptide is eight        to ten amino acids in length;    -   b. harvesting CD8⁺ cells from said subject;    -   c. stimulating said CD8⁺ cells with said APC or said nnAPC cell        line for about six to seven days;    -   d. stimulating said CD8⁺ cells with IL-2 and IL-7 in media;    -   e. mixing peripheral blood monocytes collected from said subject        with about 20 μg/ml of each peptide;    -   f. irradiating said CD8-depleted peripheral blood monocyte        suspension with about 5,000 rads of γ-radiation;    -   g. isolating adherent peripheral blood monocytes;    -   h. loading said adherent peripheral blood monocytes with about        100 ng/ml of said epitope;    -   i. combining said CD8⁺ cells with said adherent peripheral blood        monocytes at a ratio of about ten CD8⁺ cells to one peripheral        blood monocyte;    -   j. stimulating said combined suspension of CD8⁺ cells and        peripheral blood monocytes for about six to seven days;    -   k. stimulating said suspension of CD8⁺ cells and peripheral        blood monocytes with IL-2 and IL-7 in media;    -   l. assaying CD8⁺ suspension for suitable CTL activity, purity,        sterility and endotoxin content; and    -   m. inoculating said subject with CD8⁺ suspension.

Another embodiment of the present invention provides a method fortreating a subject with autoimmune disease, including, but not limitedto, rheumatoid arthritis, lupus, psoriasis, autoimmune nephritis,multiple sclerosis, insulin dependent diabetes, autoimmune thyroiditis,Crohn's disease, inflammatory bowel disease, graft versus host diseaseand transplant rejection, and/or allergic diseases, including, but notlimited to, food allergy, hay fever, allergic rhinitis, allergic asthmaand venom allergy, comprising:

-   -   a. preparing a naturally occurring antigen-presenting cell (APC)        or a non-naturally occurring antigen-presenting cell line        (nnAPC), wherein said APC or said nnAPC is capable of presenting        up to about fifteen different peptide-epitope molecules that is        associated with such diseases, preferably about ten peptides,        simultaneously where each peptide is eight to ten amino acids in        length;    -   b. harvesting CD8⁺ cells from said subject;    -   c. stimulating said CD8⁺ cells with said APC or said nnAPC cell        line for about six to seven days;    -   d. stimulating said CD8⁺ cells with IL-2 and IL-7 in media;    -   e. mixing peripheral blood monocytes collected from said subject        with about 20 μg/ml of each peptide said APC or said nnAPC can        present;    -   f. irradiating said CD8-depleted peripheral blood monocyte        suspension with about 5,000 rads of γ-radiation;    -   g. isolating adherent peripheral blood monocytes;    -   h. loading said adherent peripheral blood monocytes with about        100 ng/ml of said epitope;    -   i. combining said CD8⁺ cells with said adherent peripheral blood        monocytes at a ratio of about ten CD8⁺ cells to one peripheral        blood monocyte;    -   j. stimulating said combined suspension of CD8⁺ cells and        peripheral blood monocytes for about six to seven days;    -   k. stimulating said suspension of CD8⁺ cells and peripheral        blood monocytes with IL-2 and IL-7 in media;    -   l. assaying CD8⁺ suspension for suitable CTL activity, purity,        sterility and endotoxin content; and    -   m. inoculating said subject with CD8⁺ suspension.

Another embodiment of the present invention is a method of treatingallergic and/or autoimmune diseases wherein the nnAPC presents thefollowing peptides, SEQ ID NO:15 to SEQ ID NO: 49.

Another embodiment of the present invention is a method of treating anon-cancer disease or disease condition that results in an insufficientor inadequate immune response that is normally associated with Class IHLA molecules, wherein the treatment eliminates infected or transformedcells wherein said elimination has been demonstrated to be mediated byCTLs.

Another embodiment of the present invention is a method of treating anon-cancer disease or disease condition that results in an insufficientor inadequate immune response that is normally associated with Class IHLA molecules, wherein infected or transformed cells that have beenshown to be susceptible to elimination by CTL are treated by the methodcomprising:

-   -   a. preparing a naturally occurring antigen presenting cell (APC)        or a non-naturally occurring antigen-presenting cell line        (nnAPC), wherein said APC or said nnAPC is capable of presenting        up to about fifteen different peptide molecules that is        associated with said disease or disease condition, preferably        about ten different peptide epitope molecules, simultaneously        where each peptide is about six to twelve amino acids in length,        preferably about eight to ten amino acids in length and in a        concentration range of about 10 nM to 100 μM;    -   b. harvesting CD8⁺ cells from said subject or a suitable donor;    -   c. stimulating said CD8⁺ cells with said APC or said nnAPC cell        line;    -   d. adding said CD8⁺ cells to media that contains a cytokine,        such as, IL-2, IL-7 or CGM, preferably, IL-2, or IL-2 and IL-7        in combination;    -   e. mixing unsuspended peripheral blood monocytes, or        alternatively, CD8-depleted peripheral blood monocytes collected        from said subject or a suitable donor with about 10 to 50 μg/ml        of a peptide;    -   f. irradiating said peripheral blood monocyte suspension with a        sufficient dose of γ-radiation necessary to sterilize all        components in the suspension, except the desired peripheral        blood monocytes, such as a dose in the range of about 3,000 to        7,000 rads, preferably about 5,000 rads;    -   g. isolating adherent peripheral blood monocytes;    -   h. loading said adherent peripheral blood monocytes with about        10 ng/ml to 10 μg/ml of said each peptide;    -   i. combining said CD8⁺ cells with said adherent peripheral blood        monocytes at a ratio of about ten CD8⁺ cells to one peripheral        blood monocyte;    -   j. optionally stimulating said combined suspension of CD8⁺ cells        and peripheral blood monocytes for about six to seven days;    -   k. optionally stimulating said suspension of CD8⁺ cells and        peripheral blood monocytes with IL-2 and IL-7 in media;    -   l. optionally assaying CD8⁺ suspension for suitable CTL        activity, and optionally assaying for CTL purity, sterility and        endotoxin content; and    -   m. inoculating said subject with CD8⁺ suspension.

The present invention provides a non-naturally occurringantigen-presenting cell (nnAPC) derived from Drosophila melanogastercells transfected with DNA for expression, wherein the nnAPC is capableof simultaneously presenting up to fifteen different peptide moleculesassociated with allergic and/or autoimmune disease, preferably tenpeptide molecules that are encoded by the DNA.

The present invention provides a non-naturally occurringantigen-presenting cell (nnAPC) derived from Drosophila melanogastercells transfected with human class I HLA, binding, and co-stimulatorymolecule's DNA for expression, wherein the nnAPC is capable ofpresenting up to fifteen different peptide molecules associated withallergic and/or autoimmune disease, preferably ten peptide moleculesthat are encoded by the DNA simultaneously.

Another embodiment of the present invention provides a nnAPC thatpresents peptides that are associated with various desired functionsthat enhance the treatment of the subject. For example, in addition topeptides associated with the disease or disease condition being treated,the nnAPC can present peptides associated with accessory molecules suchas, lymphocyte function antigens (LFA-1, LFA-2 and LFA-3), intercellularadhesion molecule 1 (ICAM-1), T-cell co-stimulatory factors (CD2, CD28,B7) enhance cell-cell adhesion or transduce additional cell activationsignals.

Another embodiment of the present invention provides a nnAPC thatpresents peptides that are associated with allergic and/or autoimmunediseases. For example, the peptides associated or derived from IgE maybe presented with peptides associated or derived from an allergen, orfurther in combination with CD40L peptides.

Another embodiment of the present invention provides a method formanufacturing non-naturally occurring antigen-presenting cell (nnAPC)capable of presenting up to ten different peptide molecules associatedwith allergic and/or autoimmune disease simultaneously, said methodcomprising of the step:

-   -   a. preparing a insect cell line from Drosophila melanogaster        eggs; alternatively preparing an insect cell line, where cells        are grown for twelve days, selected with peptides, preferably        tetramers, that are capable of identifying the desired cells,        and then expanding said desired cells with OKT3 and IL-2.    -   b. growing said insect cells a media that is suitable for        growing insect cells, preferably Schneider™'s Drosophila Medium;    -   c. making a pRmHa-3 plasmid from a pRmHa-1 expression vector,        where said pRmHa-3 plasmid includes a metallothionein promoter,        metal response consensus sequences and an alcohol dehydrogenase        gene bearing a polyadenylation signal isolated from Drosophila        melanogaster;    -   d. inserting into said pRmHa-3 plasmid complementary DNA for        human class I HLA A2.1, B7.1, B7.2, ICAM-1, β-2 microglobulin        and LFA-3, wherein A2.1 can be substituted with any human class        I DNA sequence;    -   e. transfecting said insect cells with a phshneo plasmid and        said pRmHa-3 plasmid containing complementary DNA;    -   f. creating nnAPC by contacting said insect cells with CuSO₄ to        induce expression of the transfected genes in said insect cells.

Professional antigen presenting cells, such as dendritic cells andmacrophages, can be loaded with IgE peptides (Dalyot-Herman et al.(2000) supra) or IgE recombinant proteins (Paglia et al. (1996) supra)or transduced with virus encoding IgE or fragments of IgE (Yang et al.,Cellular Immunology (2000) 204:29-37). These modified professionalantigen-presenting cells can then be used to activate IgE specific CD8 Tcells and generate IgE specific CTLs in vitro. Alternatively,non-professional antigen presenting cells can also be transfected ortransduced with a number of genes that encode costimulation moleculesplus the genes that encode IgE and a fragment of IgE. The modifiednon-professional antigen presenting cells thus can be used to stimulateIgE specific CD8 T cells for generation of IgE specific CTLs.

The insect cells of the present invention are grown in a media suitablefor growing insects, hereinafter referenced to as “insect growth media”.Insect growth media are commercially available from a number of vendors,such as, Schneider™'s Drosophila Medium, Grace's Insect Media, andTC-100 Insect Media. Alternatively, insect growth media can be preparedby one of ordinary skill in the art. Typically, the media will includecomponents necessary to promote and sustain the growth of insects cells,such as, inorganic salts (for example, calcium chloride, magnesiumsulfate, potassium chloride, potassium phosphate, sodium bicarbonate,sodium chloride, and sodium phosphate), amino acids various carbohydrateand chemical species (Imogene Schneider, Exp. Zool. (1964) 156(491-104). Alternatively, the media can also include vitamins, minerals,and other components that aid in the growth of insect cells.

Following is a list of abbreviations and definitions used in the presentspecification.

ABBREVIATIONS

-   -   APC Antigen-presenting cells    -   CD8⁺ CD8⁺ T cells    -   CTL Cytotoxic T lymphocyte    -   FAS Also known as CD95, epitope on T cells    -   ICAM Intercellular adhesion molecule    -   IL Interleukin    -   LFA Lymphocyte function antigens    -   MHC Major histocompatibility complex    -   nnAPC Non-naturally occurring antigen-presenting cell    -   PBMC Peripheral blood mononuclear cell    -   PBS Phosphate-buffered saline    -   PCR Polymerase chain reaction    -   RPMI Roswell Park Memorial Institute    -   RWJPRI The R.W. Johnson Pharmaceutical Research Institute    -   T Target    -   TCR T cell antigen receptor

Following is a list of abbreviations used in the present specificationfor various peptide epitopes. The individual amino acid residues areidentified according to a single letter code that is readily known andused by those of ordinary skill in the art.

ABBREVIATIONS AMINO ACID 3-Letter 1-Letter alanine ala A valine val Vleucine leu L isoleucine ile I proline pro P phenylalanine phe Ftrytophan tyr W methionine met M glycine gly G serine ser S threoninethr T cysteine cys C tyrosine tyr Y asparagine asn N glutamine gln Qaspartic acid asp D glutamic acid glu E lysine lys K arginine arg Rhistidine his H

PEPTIDE EPITOPE ABBREVIATIONS

As used herein the term IgE 11 refers to the amino acid sequenceKPCKGTASM (SEQ ID NO: 1).

As used herein the term IgE 209 refers to the amino acid sequenceIPPSPLDLY (SEQ ID NO: 2).

As used herein the term IgE 366 refers to the amino acid sequenceGSNQGFHF (SEQ ID NO: 3).

As used herein the term IgE 29 refers to the amino acid sequenceFPNPVTVTW (SEQ ID NO: 4).

As used herein the term IgE 105 refers to the amino acid sequenceHSSCDPNAF (SEQ ID NO: 5).

As used herein the term IgE 114 refers to the amino acid sequenceHSTIQLYCF (SEQ ID NO: 6).

As used herein the term IgE 363 refers to the amino acid sequenceKSNGSNQGF (SEQ ID NO: 7).

As used herein the term IgE 307 refers to the amino acid sequenceRSAPEVYVF (SEQ ID NO: 8).

As used herein the term IgE 44 refers to the amino acid sequenceMSTVNFPAL (SEQ ID NO: 9).

As used herein the term IgE 411 refers to the amino acid sequenceTSLGNTSLR (SEQ ID NO: 10).

As used herein the term IgE 16 refers to the amino acid sequenceTASMTLGCL (SEQ ID NO: 11).

As used herein, the term IgE 159 refers to the amino acid sequence ofASTCSKLNI (SEQ ID NO: 12).

As used herein, the term IgE 125 refers to the amino acid sequence ofGHILNDVSV (SEQ ID NO: 13).

As used herein the term CD40L 17 refers to the amino acid sequenceLPASMKIFM (SEQ ID NO: 15).

As used herein the term CD40L 186 refers to the amino acid sequenceRPFIVGLWL (SEQ ID NO: 16).

As used herein the term CD40L 118 refers to the amino acid sequenceDPQIAAHVV (SEQ ID NO: 17).

As used herein the term CD40L 220 refers to the amino acid sequenceQSVHLGGVF (SEQ ID NO: 18).

As used herein the term CD40L 9 refers to the amino acid sequenceSPRSVATGL (SEQ ID NO: 19).

As used herein the term CD40L 195 refers to the amino acid sequenceKPSIGSERI (SEQ ID NO: 20).

As used herein the term CD40L 252 refers to the amino acid sequenceFSSFGLLKL (SEQ ID NO: 21).

As used herein the term CD40L 7 refers to the amino acid sequenceQPSPRSVAT (SEQ ID NO: 22).

As used herein the term CD40L 181 refers to the amino acid sequenceEPSSQRPFI (SEQ ID NO: 23).

As used herein the term CD40L 79 refers to the amino acid sequenceLSLLNCEEM (SEQ ID NO: 24).

As used herein, the term CD40L 152 refers to the amino acid sequence ofVMLENGKQL (SEQ ID NO: 25).

As used herein, the term CD40L 146 refers to the amino acid sequence ofTMKSNLVML (SEQ ID NO: 26).

As used herein, the term CD40L 235 refers to the amino acid sequence ofSVFVNVTEA (SEQ ID NO: 27).

As used herein, the term CD40L 38 refers to the amino acid sequence ofGSVLFAVYL (SEQ ID NO: 28).

As used herein, the term CD40L 19 refers to the amino acid sequence ofASMKIFMYL (SEQ ID NO: 29).

As used herein the term CD40L 24 refers to the amino acid sequenceFMYLLTVFL (SEQ ID NO: 30).

As used herein the term CD40L 167 refers to the amino acid sequenceGLYYIYAQV (SEQ ID NO: 31).

As used herein the term CD40L 22 refers to the amino acid sequenceKIFMYLLTV (SEQ ID NO: 32).

As used herein the term CD40L 36 refers to the amino acid sequenceMIGSALFAV (SEQ ID NO: 33).

As used herein the term CD40L 58 refers to the amino acid sequenceNLHEDFVFM (SEQ ID NO: 34).

As used herein the term CD40L 170 refers to the amino acid sequenceYIYAQVTFC (SEQ ID NO: 35).

As used herein the term CD40L 26 refers to the amino acid sequenceYLLTVFLIT (SEQ ID NO: 36).

As used herein the term CD40L 231 refers to the amino acid sequenceLQPGASVFV (SEQ ID NO: 37).

As used herein the term CD40L 45 refers to the amino acid sequenceYLHRRLDKI (SEQ ID NO: 38).

As used herein the term CD40L 147 refers to the amino acid sequenceTMSNNLVTL (SEQ ID NO: 39).

As used herein, the term CD40L 229 refers to the amino acid sequence ofFELQPGASV (SEQ ID NO: 40).

As used herein, the term CD40L 160 refers to the amino acid sequence ofQLTVKRQGL (SEQ ID NO: 41).

As used herein, the term CD40L 35 refers to the amino acid sequence ofQMIGSALFA (SEQ JD NO: 42).

As used herein, the term CD40L 185 refers to the amino acid sequence ofSQAPFIASL (SEQ ID NO: 43).

As used herein, the term CD40L 19 refers to the amino acid sequence ofISMKIFMYL (SEQ ID NO: 44).

As used herein, the term CD40L 153 refers to the amino acid sequence ofVTLENGKQL (SEQ ID NO: 45).

As used herein, the term CD40L 126 refers to the amino acid sequence ofVISEASSKT (SEQ ID NO: 46).

As used herein, the term CD40L 227 refers to the amino acid sequence ofGVFELQPGA (SEQ ID NO: 47).

As used herein, the term CD40L 20 refers to the amino acid sequence ofSMKIFMYLL (SEQ ID NO: 48).

As used herein, the term CD40L 165 refers to the amino acid sequence ofRQGLYYIYA (SEQ ID NO: 49).

As used herein, the term IgE 47 refers to the amino acid sequence ofSLNGTTMTL NO: 50).

As used herein, the term IgE 96 refers to the amino acid sequence ofWVDNKTFSV (SEQ ID NO: 51).

As used herein, the term IgE 185 refers to the amino acid sequence ofWLSDRTYTC (SEQ ID NO: 52).

As used herein, the term IgE 309 refers to the amino acid sequence ofALSDRTYTC (SEQ ID NO: 53).

As used herein, the term IgE 876 refers to the amino acid sequence ofSLLTVSGAWA (SEQ ID NO: 54).

As used herein, the term IgE 883 refers to the amino acid sequence ofWLEDGQVMDV (SEQ ID NO: 55).

As used herein, the term IgE 884 refers to the amino acid sequence ofTLTVTSTLPV (SEQ ID NO: 56).

As used herein, the term IgE 887 refers to the amino acid sequence ofQMFTCRVAHT (SEQ ID NO: 57).

As used herein, the term IgE 890 refers to the amino acid sequence ofYATISLLTV (SEQ ID NO: 58).

As used herein, the term IgE 895 refers to the amino acid sequence ofTLACLIQNFM (SEQ ID NO: 59).

As used herein, the term IgE 898 refers to the amino acid sequence ofQVMDVDLSTA (SEQ ID NO: 60).

TERMS AND DEFINITIONS

As used herein, the term “adoptive immunotherapy” refers theadministration of donor or autologous T lymphocytes for the treatment ofa disease or disease condition, wherein the disease or disease conditionresults in an insufficient or inadequate immune response that isnormally associated with Class I HLA molecules. Adoptive immunotherapyis an appropriate treatment for any disease or disease condition wherethe elimination of infected or transformed cells has been demonstratedto be achieved by CTLs. For example, disease or disease conditionsinclude but are not limited to cancer and/or tumors, such as, melanoma,prostate, breast, colo-rectal, stomach, throat and neck, pancreatic,cervical, ovarian, bone, leukemia and lung cancer; viral infections,such as, hepatitis B, hepatitis C, human immunodeficiency virus; andbacterial infections, such as, malaria; tuberculosis, and lysteriamonocytogenesis.

As used herein, the term “B7.1” refers to a co-stimulatory moleculeassociated with antigen-presenting cells.

As used herein, the term “BCNU” refers to carmustine, also known as,1,3-bis (2-chloroethyl)-1-nitrosourea.

As used herein, the term “BSE” refers to bovine spongiform encephalitis.

As used herein, the term “CD” refers to clusters of differentiation, Tlymphocytes (originally), B lymphocytes, monocytes, macrophages, andgranulocytes grouped by antigen epitopes and function.

As used herein, the term “DTIC” refers to dacarbazine,5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide.

As used herein, the term “ex vivo” or “ex vivo therapy” refers to atherapy where biological materials, typically cells, are obtained from apatient or a suitable alternate source, such as, a suitable donor, andare modified, such that the modified cells can be used to treat apathological condition which will be improved by the long-term orconstant delivery of the therapeutic benefit produced by the modifiedcells. Treatment includes the re-introduction of the modified biologicalmaterials, obtained from either the patient or from the alternatesource, into the patient. A benefit of ex vivo therapy is the ability toprovide the patient the benefit of the treatment, without exposing thepatient to undesired collateral effects from the treatment. For example,cytokines are often administered to patients with cancer or viralinfections to stimulate expansion of the patient's CTLs. However,cytokines often cause the onset of flu like symptoms in the patients. Inan ex vivo procedure, cytokines are used to stimulate expansion of theCTLs outside of the patient's body, and the patient is spared theexposure and the consequent side effects of the cytokines. Alternativelyunder suitable situations, or conditions, where appropriate and wherethe subject can derive benefit, the subject can be treated concurrentlywith low level dosages of a interferon.

As used herein, the term “HEPES” refers toN-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid buffer.

As used herein, the term “HLA-A2.1” refers to a HLA Class I moleculefound in approximately 45% of Caucasians.

As used herein, the term “MPC-10” refers to a magnetic particleconcentrator.

As used herein, the term “NK cells” refers to natural killer cells.

As used herein, the term “OKT3” refers to ORTHOCLONE OKT3,muromonab-CD3, anti-CD3 monoclonal antibody.

As used herein, the term “TAP-1, 2” refers to Transporter Associatedwith Antigen Processing-1, 2.

As used herein, the term “Th cells” refers to Helper T cells, CD4⁺.

As used herein, the term “C-lectin” refers to a peptide of the sequencethat has been found to be associated with ovarian cancer.

As used herein, the term “major histocompatibility complex” or “MHC” isa generic designation meant to encompass the histocompatibility antigensystems described in different species including the human leucocyteantigens (HLA).

As used herein, the terms “epitope,” “peptide epitope,” “antigenicpeptide” and “immunogenic peptide” refers to a peptide derived from anantigen capable of causing a cellular immune response in a mammal. Suchpeptides may also be reactive with antibodies from an animal immunizedwith the peptides. Such peptides may be about five to twenty amino acidin length preferably about eight to fifteen amino acids in length, andmost preferably about nine to ten amino acids in length.

As used herein, the term “analog” includes any polypeptide having anamino acid residue sequence substantially identical to the polypeptidesequence of the present invention in which one or more residues havebeen conservatively substituted with a functionally similar residue andwhich displays the functional aspects of the present invention asdescribed herein. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid or another.

As used herein, the term “conservative substitution” also includes theuse of a chemically derivatized residue in place of a non-derivatizedresidue.

As used herein, the term “chemical derivative” refers to a subjectpolypeptide having one or more residues chemically derivatized byreaction of a functional side group. Examples of such derivatizedmolecules include for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those proteins or peptides which contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Proteins or polypeptides of thepresent invention also include any polypeptide having one or moreadditions and/or deletions or residues relative to the sequence of apolypeptide whose sequence is encoded is the corresponding nucleicsequence of the present invention, so long as the requisite activity ismaintained.

Cytolytic T cells (CD8⁺) are the main line of defense against viralinfections. CD8⁺ lymphocytes specifically recognize and kill host cellsthat are infected by a virus. Theoretically, it should be possible toharness the immune system to combat other types of diseases includingcancer. However, few in vitro/ex vivo procedures have been available forspecifically activating CTLs. The identification of key allergic and/orautoimmune antigens noted herein and a method for specific in vitroactivation CTLs described below now allow testing of the concept ofadoptive immunotherapy of allergic and/or autoimmune diseases.

All naive T cells require two signals for activation to elicit an immuneresponse. For CD8⁺ lymphocytes (CTLs), the first signal, which impartsspecificity, consists of presentation to the CD8⁺ cell of an immunogenicpeptide fragment (epitope) of the antigen bound to the Class I MHC (HLA)complex present on the surface of antigen-presenting cells (APCs). Thiscomplex is recognized specifically by a T cell antigen receptor (TCR),which communicates the signal intracellularly.

Binding to the T cell receptor is necessary but not sufficient to induceT cell activation, and usually will not lead to cell proliferation orcytokine secretion. Complete activation requires a second co-stimulatorysignal(s), these signals serve to further enhance the activationcascade. Among the co-stimulatory molecules on antigen-presenting cells,B7 and cell adhesion molecules (integrins) such as ICAM-1 assist in thisprocess by binding to CD28 and LFA-1, respectively, on the T cell. Whena CD8⁺ cell interacts with an antigen-presenting cell bearing animmunogenic peptide (epitope) bound by a Class I MHC molecule in thepresence of appropriate co-stimulatory molecule interactions, the CD8⁺cell becomes a fully activated cytolytic T cell.

Lymphocyte-mediated cell killing involves a sequence of biologicalevents beginning with the binding of the CD8⁺ CTL to an antigen-bearingtarget (tumor) cell by means of the recognition process described abovefor T cell activation. The interaction begins with the binding ofantigen in association with an MHC Class I molecule on the APC or targetcell to the T cell antigen receptor (TCR). Accessory molecules such aslymphocyte function antigens (LFA-1, LFA-2 and LFA-3), intercellularadhesion molecule 1 (ICAM-1), T cell co-stimulatory factors (CD2, CD28,B7) enhance cell-cell adhesion or transduce additional cell activationsignals.

After cell-cell interaction, the CTL kills the target cell through theaction of soluble cytolytic mediators (perforin and granzymes stored incytoplasmic granules in the T cell) and a CTL surface molecule (FASligand). After the cytolytic attack, target cells die by necrosis(membrane perforation and organelle destruction) or apoptosis (chromatincondensation, DNA fragmentation and membrane blebbing).

The mechanisms of lymphocyte-mediated cytolysis is graphically depictedin FIG. 2. In Panel A of FIG. 2, after binding to the target cell,cytoplasmic granules in the CTL are rapidly reoriented toward the targetcell for release of granules containing perforin and granzymes into theintercellular space. These proteolytic enzymes form pores in the plasmamembrane of the target cell eventually leading to cell necrosis. InPanel B, after binding to the target cell, the level of FAS expressionon the CTL increases. The interaction of FAS and the FAS receptor on thetarget cell leads to apoptosis. Proteases such as CPP32 and othersrelated to IL-1b-converting enzyme (ICE) have been implicated in theinduction of apoptosis.

It is possible to use naturally-occurring antigen-presenting cells, forexample, dendritic cells, macrophages, autologous tumor cells for invitro CD8⁺ activation. However, the efficiency of activation followingthis approach is low. This is because the Class I molecules of nativeAPCs contain many other types of peptide epitopes besides tumorepitopes. Most of the peptides are derived from normal innocuous cellproteins, resulting in a dilution of the number of active native APCsthat would actually be effective against a tumor (Allison et al., Curr.Op. Immunol. (1995) 7:682-686).

A more direct and efficient approach to this problem is to specificallyactivate CD8⁺ cells only with those epitopes relevant to combating aspecific disease, (such as allergic and/or autoimmune disease). To thisend, an artificial antigen presenting cell is created by expressing MHCClass I molecules in Drosophila melanogaster (fruit fly) cells. SinceDrosophila does not have an immune system, the TAP-1,2 peptidetransporters involved in loading peptide epitopes onto class I moleculesare absent. As a result, the class I molecules appear on the Drosophilacell surface as empty vessels. By incubating these transfectedDrosophila cells with exogenous peptides that bind to the class Imolecules, such as, cancer or tumor specific epitopes, including butlimited to, melanoma specific epitopes, it is possible to occupy everyclass I molecule with the same peptide. High density expression of classI molecules containing a single peptide in these Drosophila APCs permitgeneration of cytotoxic CD8⁺ T cells in vitro which are completelyspecific for the antigen peptide. Methods and procedures for preparingDrosophila cells are taught in U.S. Pat. No. 5,529,921 issued Jun. 25,1996 entitled “In Vitro Activation of Cytotoxic T-Cells Using InsectCells Expressing Human Class I MHC and β2-Microglobulin”, and U.S. Pat.No. 5,314,813 issued May 24, 1994 entitled “Drosophila Cell LinesExpressing Genes Encoding MHC Class I Antigens And β2-Microglobulin andCapable of Assembling Empty Complexes and Methods of Making Said CellLines”. In particular, U.S. Pat. No. 5,529,921 discloses at column 26,line 56 to column 28, line 22 various methods of separating out and/orenriching cultures of precursor cells.

Additionally, this feature eliminates the need for in vivo stimulationof the immune system with various cytokines. Thereby resulting in atreatment that fore goes the side effects caused by cytokines.Alternatively under suitable situations, or conditions, whereappropriate and where the subject can derive benefit, the subject can betreated concurrently with low level dosages of α interferon.

Eliminating the need for in vivo stimulation with cytokines provides animprovement to the quality of patient care. Treatment regimes thatinclude the administration of cytokines to patients often result in thepatient developing flu-like symptoms, such as nausea, vomiting, andfever. These side reactions are generally not life threatening, althougha particularly severe reaction occurring in a patient who is already ina weaken condition could result in a life endangering situation. Anotherconsideration is the adverse impact such side reactions have on patientacceptance and compliance of an otherwise beneficial treatment regime.Removing the need for in vivo stimulation with cytokines results in atreatment regime that improves the comfort of the patient, and providesthe clinician with an effective method of treatment that his or herpatient is more likely to comply with.

The utility of this method for adoptive immunotherapy has beendemonstrated in mice using transfected Drosophila cells as APCs and CD8⁺cells from the 2C line of T cell receptor (TCR) transgenic mice. In thissystem, purified CD8⁺ 2C cells are highly responsive to in vitropeptides presented by MHC Class I (L^(d))-transfected Drosophila cellsalso bearing the co-stimulatory molecules B7-1 and ICAM-1. TransfectedDrosophila cells as a probe for defining the minimal requirements forstimulating unprimed CD8+ T cells (Cai et al., P.N.A.S .USA (1996)93:14736-14741). Alternatively, when un-separated mouse spleen cells areused as responders in place of purified 2C cells, the need forco-stimulatory molecules does not apply. In this instance, the CD8⁺cells in the spleen population receive “bystander” co-stimulation fromactivated B cells. Utilizing this finding, it has been possible to showthat MHC Class I (L^(d))-transfected Drosophila cells are able to inducenormal DBA/2 mouse spleen cells to respond to syngeneic P815 mastocytomatumor-specific peptides in vitro in the absence of added lymphokines.Injection of these CTLs into DBA/2 mice bearing P815 mastocytoma led torapid tumor regression (Sun et al., Immunity (1996) 4:555-564).

The use of any natural, or artificial, antigen presenting cell (APC)system to generate cytotoxic T lymphocytes in vitro is limited by theantigen specificities these systems are capable of generating.

The following APC systems have been utilized to generateantigen-specific CTL's to single epitopes:

-   -   1. Human dendritic cells (DC) pulsed with defined peptides;    -   2. Peripheral blood mononuclear cells (PBMCs) which have been        driven to lymphoblasts and pulsed with peptides;    -   3. Lymphoblastoid cell lines (LCL) where the natural peptides        are acid-stripped and loaded with the peptides of interest;    -   4. Drosophila cells engineered to express empty class I        molecules; and Mouse 3T3 cells transfected with human class I        and co-stimulatory molecules. (J-B. Latouche and M. Sadelain,        Nature Biotech (2000) 18:405-409).

Dendritic cells (DCs) are considered the primary antigen presenting cellsystem in humans because of their wide application in presenting primaryantigen cells. Self or foreign proteins are processed within a DC. Theresultant peptide epitopes are presented by HLA molecules, and aretransported to the surface of the DC. However, it was found that DCswould not consistently generate in vitro, CTLs directed against fourdifferent peptides. This would have provided CTLs having activitycorresponding to each of the four peptides. In addition, it was alsofound that the phenotype of the DC at the time of peptide pulsing,mature or immature, did not effect the outcome.

Alternatively, Drosophila cell stimulation usually resulted in CTLsdirected against up to ten different types of peptides. This providesCTLs that are active to each of the ten peptides.

The ability of Drosophila cells and DC to elicit CTL responses wereevaluated, initially to a single peptide epitope, following the standardstimulation protocols for each, in order to compare DCs and transfectedDrosophila cells. Immature DCs were generated by culturing for one weekautologous monocytes in the presence of IL-4 and GM-CSF. Mature DCs wereobtained from immature DCs by addition of TNF α to the culture mediumtwenty-four hours prior to harvesting. DCs (immature and mature) wereharvested, pulsed with peptides and mixed with purified CD8 cellsfollowing the procedure used for the stimulation of CD8 cells andpeptide-pulsed Drosophila cells. Drosophila cells were found to begenerally better stimulators than DC. Further, DCs displaying either theimmature or mature phenotype were not as efficient as Drosophila cellsin eliciting specific CTL responses when defined peptides were used topulse the APCs. This is particularly surprising, because of the dominantrole played by DCs in the immune system.

Preparation of Cytotoxic Lymphocytes

CD8⁺ cells isolated from leukapheresis samples by positive selectionwith anti-CD8 antibody are stimulated against IgE and/or CD40Lassociated peptides presented by Drosophila cells expressing Human ClassI molecules (HLA-A2.1), B7.1, ICAM-1, LFA-3 and B7.2. CD8⁺ cells arere-stimulated for two rounds with autologous monocytes loaded with thepeptide epitope in the presence of IL-2 and IL-7. CTLs arenon-specifically expanded with OKT3 and IL-2. CTL activity is measuredagainst cells and purity of CD8⁺ T cells is assessed by flow cytometry.

The manufacturing processes and protocols are done according to GoodLaboratory Practices and Good Manufacturing Practices. “Good LaboratoryPractices” and “Good Manufacturing Practices” are standards oflaboratory and manufacturing practices, which are set by United StatesFood and Drug Administration, and are readily known to those of skill inthe art. The CTLs are monitored for identity, viability, CTL activity,sterility, and endotoxin content.

The following examples are intended to illustrate but not limit thepresent invention.

Example 1 Manufacture of Drosophila Antigen-Presenting Cells

The Schneider S2 cell line was prepared from Drosophila melanogaster(Oregon-R) eggs according to published procedures and has been depositedwith the American Type Culture Collection (CRL 10974). S2 cells aregrown in commercial Schneider's Drosophila medium supplemented with 10%fetal bovine serum.

The pRmHa-3 plasmid vector for expressing MHC Class I and co-stimulatoryproteins in S2 cells was derived from the pRmHa-1 expression vectorconstructed as described in the literature. It contains ametallothionein promoter, metal response consensus sequences and analcohol dehydrogenase gene bearing a polyadenylation signal isolatedfrom Drosophila melanogaster.

Complementary DNAs for Transfection were Prepared as Follows:

-   HLA-A2.1 and β-2 microglobulin: Reverse transcription-PCR from K562    cells using primers derived from the published sequence.-   B7.1: Reverse transcription-PCR from K562 cells using primers    derived from the published sequence.-   ICAM-1: Reverse transcription-PCR from K562 cells using primers    derived from the published sequence.-   B7.2: Reverse transcription-PCR from HL-60 cells (ATCC CCL-240)    using primers derived from the published sequence.-   LFA-3: Reverse transcription-PCR from HL-60 cells (ATCC CCL-240)    using primers derived from the published sequence.

Complementary DNAs were individually inserted into the pRmHa-3 vector.S2 cells were transfected with a mixture of HLA-A2.1, B7.1 and ICAM-1plasmid DNAs and the phshneo plasmid using the calcium phosphateprecipitation method. Stably transfected cells were selected byculturing in Schneider's medium containing geneticin. Twenty-four hoursbefore use, expression of the transfected genes was induced by additionof CuSO₄. The level of expression was assessed by flow cytometry usinganti-HLA-A2.1, anti-B7.1 and anti-ICAM-1 antibodies. HLA expression bygreater than 30% of the cells is necessary for efficient in vitroactivation of CD8⁺ lymphocytes.

Isolation of Human CD8⁺ Cells

CD8⁺ cells are isolated from leukapheresis samples by positive selectionusing the Dynabeads™ isolation procedure (Dynal). An anti-human CD8mouse monoclonal antibody (50 μg/ml in human gamma globulin[Gammagard®]) is added to washed cells in Dulbecco's PBS supplementedwith 1% human serum albumin (Baxter-Hyland) and 0.2% Na citrate. Afterincubation at 4° C. for forty-five minutes with gentle mixing, the cellsare washed and re-suspended in the same buffer containing Dynal magneticbeads (Dynabeads™) coated with sheep anti-mouse IgG at a bead to cellratio of 1:1. The cells and beads are placed into a sterile tube andgently mixed at 4° C. for forty-five minutes. At the end of this time,the antibody-bound cells are removed magnetically using the MPC-1®separator according to the manufacturer's instructions (Dynal).Dissociation of the CD8 cell-bead complex is achieved by incubation at37° C. for forty-five minutes in the presence of CD8 peptide₅₉₋₇₀(AAEGLDTQRFSG, SEQ. ID. NO.:52). Free beads are removed magnetically andthe CD8 cells are counted and analyzed by flow cytometry to evaluatepurity. Recovery of CD8⁺ cells is typically greater than 80%. Table 1summarizes the cell composition of fourteen separate CD8⁺ preparationsfrom normal human PBMC preparations by positive selection with anti-CD8antibody.

TABLE 1 Purification of CD8⁺ Cells by Positive Selection Analyzed byFlow Cytometry PBMC POST SELECTION CELL TYPE Mean % (Range) Mean %(Range) CD8 T cells 15% (7-24) 82%   (56-95) CD4 T cells 36% (14-52)  2% (0.1-10) CD 14 Monocytes 15% (7-26) 0.8%  (0.2-2) CD15 Neutrophils 12%(8-21) 0.6%  (0.1-3) CD19 B cells  2% (0.4-7)   3% (0.5-9) CD56 NK cells 6% (2-17) 6%  (0.1-20)

In Vitro Immunization of Purified Human CD8⁺ Cells

Primary Stimulation Transfected Drosophila S2 cells are incubated inSchneider's medium (10⁶ cells/ml) supplemented with 10% fetal calf serumand CuSO₄ at 27° C. for twenty-four hours. Cells are harvested, washedand re-suspended in Insect X-press medium (BioWhittaker) containing 100μg/ml human tyrosinase₃₆₉₋₃₇₇ (RWJPRI). Following incubation at 27° C.for three hours, the S2 cells are mixed with CD8⁺ cells at a ratio of1:10 in RPMI medium (Gibco) supplemented with 10% autologous serum. Thecell mixture is incubated for four days at 37° C. during which theDrosophila cells die off. On Day 5, IL-2 (20 U/ml) and IL-7 (30 U/ml)are added with a media change to selectively expand thetyrosinase-specific CTL population.

Re-stimulation: Frozen, autologous, CD8-depleted PBMCs, obtained at thetime of leukapheresis, are thawed, washed and re-suspended at 10⁶cells/ml in RPMI medium containing 10% autologous serum (as a source ofβ2 microglobulin) and 20 μg/ml of peptide epitope. Followingγ-irradiation (5,000 rads), the cells are incubated at 37° C. for twohours. Non-adherent cells are removed by washing with Dulbecco's PBS.Adherent monocytes are loaded with the tyrosinase epitope by incubationfor 90 minutes in Hepes-buffered RPMI medium containing 10% autologousserum and 10 μg/ml of peptide epitope. The supernatant is removed andthe Drosophila-activated CD8⁺ cell suspension (3×10⁶ cells/ml in RPMImedium with 10% autologous serum) is added at a ratio of ten CD8⁺ cellsto one adherent monocyte. After three to four days of culture at 37° C.,IL-2 (20 U/ml) and IL-7 (30 U/ml) are added with a medium change toselectively expand the epitope-specific CTL population.

Non-specific Expansion: CD8's non-specifically expanded and culturingthem in RPMI medium supplemented with autologous serum, anti-CD3monoclonal antibody (OKT®3), IL-2 and γ irradiated autologous PBMCs.

Assays for Activity and Purity

CTL Assay: Epitope-bearing (target) cells are used as target cells in a⁵¹Cr release assay. 5×10⁶ target cells in RPMI medium containing 4%fetal calf serum, 1% HEPES buffer and 0.25% gentamycin are labeled at37° C. for one hour with 0.1 mCi ⁵¹Cr. Cells are washed four times anddiluted to 10⁵ cells/ml in RPMI with 10% fetal bovine serum (HyClone).In a 96-well microtiter plate, 100 μl effector CTLs and 100 μlpeptide-loaded, ⁵¹Cr-labeled target cells are combined at ratios of100:1, 20:1 and 4:1 (effector:target). K562 cells are added at a ratioof 20:1 (K562) to reduce natural killer cell background lysis.Non-specific lysis is assessed using cells labeled with ⁵¹Cr asdescribed above, but not bearing the epitope cell line. Controls tomeasure spontaneous release and maximum release of ⁵¹Cr are included induplicate. After incubation at 37° C. for six hours, the plates arecentrifuged and the supernatants counted to measure ⁵¹Cr release.Percent specific lysis is calculated using the following equation:

$\frac{{{cpm}\mspace{14mu} {sample}} - {{cpm}\mspace{14mu} {spontaneous}\mspace{14mu} {release}}}{{{cpm}\mspace{14mu} {maximum}\mspace{14mu} {release}} - {{cpm}\mspace{14mu} {spontaneous}\mspace{14mu} {release}}} \times 100$

Flow Cytometry: CD8⁺ cells, before and after in vitro activation, wereanalyzed for a number of cell surface markers using fluorescentmonoclonal antibodies and FACS analysis. Results from a typicalactivation protocol using cells from a healthy donor is shown in Table2.

TABLE 2 Flow Cytometry Analysis of In Vitro Activated CD8⁺ Cells PRE-POST- ACTIVATION ACTIVATION MARKER/CELL TYPE Mean % Mean % CD8 T cell 9899 TCRαβ T cell receptor 98 92 CD 44 lymph node homing receptor 91 99CD45RO memory T cell 58 88 CD45RA 41 31 CD62L HEV homing receptor 24 38CD56 NK cell 1 11 CD25 activated T cell 0.1 29

In addition to activity and purity, CTL preparations will be assayed forsterility and endotoxin content.

REAGENTS REAGENT SUPPLIER GRADE NOTES Rh IL-2 Chiron USP sterilesolution Rh IL-7 Genzyme Research lyophilized, sterile solution PeptideRWJPRI Research Dynabeads ® M-450 Dynal GMP sheep anti-mouse IgGmagnetic beads Human serum albumin Baxter USP sterile, non-pyrogenichepatitis virus-free, 25% solution Fetal bovine serum Gemini Researchsterile, BSE-, endotoxin- mycoplasma-free Gammagard ® Baxter USPsterile, human immune globulin solution for injection Anti-CD8 antibodyRWJPRI Research mouse anti-human CD8 monoclonal antibody CD8peptide₅₉₋₇₀ RWJPRI Research release of CD8⁺ cells from magnetic beadsW6/32 ATCC Research mouse anti-human HLA-A, B, C monoclonal antibody

CELL LINES CELL LINE SUPPLIER NOTES Drosophila S2 ATCC CRL 10974 M14UCSD HLA-A2.1 human melanoma K562 ATCC Human erythroleukemic cell line;target for NK cells JY cells ATCC EBV-transformed, human B cell lineexpressing HLA-A2.1 and B7 P815 and P1024 ATCC DBA/2 mouse mastocytomacell lines Jurkat A2.1 ATCC acute T cell leukemia transfected with humanHLA-A2.1 ATCC: American Type Culture Collection

Example 2 Trial of Cytotoxic T Cell Infusions Against IgE ProducingCells Purpose of Trial

This example teaches the effectiveness of cytotoxic T Cell infusions inthe treatment of allergic diseases as assessed according to thefollowing factors:

-   -   1. Safety and toleration of re-infused autologous CTLs after in        vitro immunization;    -   2. Kinetics of infused CTLs in the systemic circulation        factoring in limiting dilution analysis;    -   3. Whole body disposition of CTLs by radioscintigraphy;    -   4. Cell composition of biopsied nodules by immunohistology        (CTLs, TH, NK, B cells); and    -   5. Regression of measurable lesions and duration of response        over two months.        Treatment with Ex Vivo Generated Autologous CTLs

All patients will receive, at least, a single infusion of autologousCTLs. The number of cycles and the dose of cells administered to eachpatient are summarized in Table 1. The number of cells generated invitro is dependent on patient-related factors such as the numbers ofPBMCs isolated from the aphaeresis procedure and the number of CD8⁺ Tcells present in each PBMC preparation. Since all of the cells generatedin vitro are re-infused into the donor, doses administered to eachpatient are necessarily varied. In an attempt to normalize the dosesbetween patients, a calculated “potency” score is recorded for eachdose. The value is obtained by multiplying the total number of cells bythe lytic activity obtained with peptide-loaded target cells. Patientsare entered into a second, third or fourth cycle of treatment based ontheir clinical status at the end of each cycle. The total number ofnaïve CD8⁺ T cells isolated is dependent on its percentage in each ofthe PBMC preparations. The percent of CD8⁺ T cells varies among thepatients. The procedure for generating CTLs ex vivo is taught in theSpecification and Example 1, above.

Up-Regulation of Class I and Melanoma-Associated Antigens in Response toIFNα-2b

In an attempt to enhance the ability of the antigen-specific CTLs tolyse IgE producing cells in vivo, low dose IFNα-2b is administered forfive consecutive days prior to the CTL infusion, and thrice weekly foran additional four weeks. One way to measure an in vivo response to thecytokine is to evaluate biopsies obtained at serial time points byimmunohistochemical analysis for positive staining with specificantibodies.

Antigenic Specificity of Ex Vivo-Generated CTLs

CTLs generated from all patients are evaluated on the day of releaseagainst peptide-loaded T2 targets, an HLA-A2 IgE producing M-14 clone 4cell line and an autologous M-14 cell line, if biopsy material wasavailable to establish a line. Each prepared dose of cells is evaluatedfor its cytolytic activity. Peptide-loaded T2 cells, presenting eithereach peptide alone, or all peptides simultaneously, are used todetermine the specificity of the CTL response generated for eachpatient. The ability to lyse endogenously-expressed, HLA-A2-associated,antigen-bearing cells is assessed with an HLA-A2 matched line or anautologous cell line. In addition to cytolytic activity,antigen-specificity is evaluated with an established method fordetecting intracellular gamma interferon production, made in response toa specific peptide stimulus. The CTLs generated at the end of the exvivo protocol are evaluated by this method. The percent of cellsspecific for each of the peptides is recorded individually. The totalnumber of specific cells in each bulk CD8 culture from a patient iscalculated by adding each of the peptide specificities detected in thatpopulation of T cells. An increase in the total number of specific cellsis detected with each successive treatment cycle.

Presence of Anergic State Did not Preclude Ability to Generate CTLs orPrevent a Clinical Response

Most of the patients treated under this protocol receive previousmedical intervention. A pretreatment skin test is performed to determineif an anergic response to a panel of seven common antigens correlateswith either an inability to generate CTLs ex vivo, or prevent adocumented clinical response. The ability to generate CTLs ex vivo doesnot correlate with the patient's pretreatment skin test results.

Example 3

IgE plays an essential role in the pathogenesis of allergic asthma.Here, we show that cytotoxic T lymphocytes (CTLs) specific for antigenicpeptides derived from IgE molecule can be generated in vitro bystimulating resting naive CD8 T cells with IgE peptides presented byartificial antigen presenting cells. The IgE specific CTLs lyse thetarget cells loaded with IgE peptides in vitro and inhibit antigenspecific IgE response in vivo. In addition, adoptive transfer of the IgEspecific CTL to an asthmatic mouse model can inhibit the development oflung inflammation and airway hypersensitivity. Thus, IgE specific CTLmay provide a treatment for allergic asthma and other IgE-mediatedallergic diseases.

Cytotoxic T lymphocytes are derived from resting naïve CD8 T cells. Inthe present of antigens and co-stimulations, resting naïve CD8 T cellscan be activated and differentiated into armed cytotoxic T cells, whichcan destroy the target cells that express the antigens. CTLs play anessential role in immunity against virus and intracellular pathogens bylysis the infected cells and/or through the effect of cytokines CTLproduced.

Identification of Antigenic Peptides from IgE Protein Sequence:

Two alleles of mouse IgE (IgE^(a) and IgE^(b)) have been describedpreviously (P06336). The alignment of the amino acid sequences of theIgE^(a) and IgE^(b) shown that 95% of the amino acid sequences areidentical. A fourteen amino acids differences are located at thejunction region between CH1 and CH2 region and another five amino aciddifferences are located at the junction region between the CH3 and CH4region. The amino acid sequence of IgE^(b) was analyzed for 9 merpeptide sequences that contain binding motifs for L^(d) and D^(b) MHCclass I molecules by using the software of the Bioinformatics &Molecular Analysis Section available athttp://bimas.dcrt.nih.gov/molbio/hla_bind/. This program ranks potentialnonapeptides based on a predicted half-time of dissociation to MHC classI molecules. Based on the ranking analysis, eight peptides with L^(d)binding motifs and five peptides with D^(b) binding motifs were selectedfor synthesis (Table 1).

The binding capacity of these synthetic peptides to L^(d) and D^(b)class I molecules were tested in an MHC class I stabilization assay (Caiet al. (1996) supra). Antigen-transporting deficient (TAP⁻) RMAS cells(H-2^(b)) or L^(d) transfected RMAS (RMAS-L^(d)) cells were cultured inthe presence of a titrated concentration of peptides at 27° C. Afterovernight culturing at 27° C., these cells were further cultured for twohours at 37° C. and the surface expression of L^(d) or D^(b) on thecells were analyzed by flow cytometry. As shown in Table 1, two IgEpeptides, IgE 11 and IgE366 bind to L^(d) strongly, whereas IgE 114binds L^(d) weakly. Of the five peptides predicted bind to D^(b), onlyIgE44 binds D^(b) strongly and two peptides, IgE16 and IgE125, bindD^(b) weakly. Interestingly, IgE366 originally predicted binding L^(d)binds both L^(d) and D^(b). Thus, a total of six peptides wereidentified that bind to either L^(d) or D^(b) MHC class I molecules.

TABLE 1 Mouse IgE^(a) amino acid sequence: SEQ ID NO: 14 1 sirnpqlyplkpckgtasmt lgclvkdyep npvtvtwysd slnmstvnfp 51 algselkvtt sqvtswgksaknftchvthp psfnesrtil vrpvnitept 101 lellhsscdp nafhstiqly cfiyghilndvsvswlmddr eitdtlaqtv 151 likeegklas tcsklniteq qwmsestftc kvtsqgvdylahtrrcpdhe 201 prgvitylip pspldlyqng apkltclvvd leseknvnvt wnqekktsvs251 asqwytkhhn nattsitsil pvvakdwieg ygyqcivdhp dfpkpivrsi 301tktpgqrsap evyvfpppee esedkrtltc liqnffpedi svqwlgdgkl 351 isnsqhstttplksngsnqg ffifsrleva ktlwtqrkqf tcqvihealq 401 kprklektis tslgntslpr sIdentification of Antigenic Peptides of Mouse IgE Sequence Peptide MHCPeptide Identification Stabilization name Selected sequence NumberScore^(a) of MHC^(b) IgE 11 L^(d) KPCKGTASM SEQ ID NO: 1 195 ++ IgE 209L^(d) IPPSPLDLY SEQ ID NO: 2 90 − IgE 366 L^(d) GSNQGFFIF SEQ ID NO: 365 ++^(c) IgE 29 L^(d) FPNPVTVTW SEQ ID NO: 4 60 − IgE 105 L^(d)HSSCDPNAF SEQ ID NO: 5 50 − IgE 114 L^(d) HSTIQLYCF SEQ ID NO: 6 50 +IgE 363 L^(d) KSNGSNQGF SEQ ID NO: 7 50 − IgE 307 L^(d) RSAPEVYVF SEQ IDNO: 8 50 − IgE 44 D^(b) MSTVNFPAL SEQ ID NO: 9 937 ++ IgE 411 D^(b)TSLGNTSLR SEQ ID NO: 10 44 − IgE 16 D^(b) TASMTLGCL SEQ ID NO: 11 22 +IgE 159 D^(b) ASTCSKLNI SEQ ID NO: 12 19 − IgE 125 D^(b) GHILNDVSV SEQID NO: 13 30 + ^(a)Calculated score in arbitrary units. ^(b)The ratio offluorescence intensity with peptides - without peptide/without peptidesless than two-fold is scored as “+’ and more than two fold is calculateas “++”. ^(c)IgE 366 also stabilizes D^(b) class I molecules.

Generation of IgE Peptide Specific CTLs In Vitro

The ability of these IgE peptides in eliciting CTL responses wasevaluated in vitro. As previously described, Drosophila cellstransfected with MHC class I plus B7-1 and ICAM-1 are potent antigenpresenting cells (APC) in activation of resting naïve CD8 T cells invitro. Resting naïve CD8 T cells were purified from mouse lymph nodesand cultured with peptide loaded Drosophila cells transfected with L^(d)or D^(b) plus B7-1 and ICAM-1 in the absence of cytokines. IL-2 (20units/ml) was added at Day 3 and every other day thereafter. The CTLactivity towards peptides loaded RMAS (K^(b), D^(b)) cells or RMAS-L^(d)cells were measured on Day 9. As shown in FIG. 1, CTLs induced by IgE 44peptide specifically lysed the RMAS cells loaded with IgE 44 peptides,neither the target cells alone nor the target cells loaded with otherIgE peptides were recognized by the IgE44 specific CTLs.

No specific CTL activity was induced by IgE16 or IgE 125 peptides, whichhave been show to bind D^(b). IgE366 was originally identified as L^(d)binding peptide, interestingly, in addition to inducing L^(d) restrictedCTLs by IgE366, IgE366 also induce D^(b) restricted CTLs (FIG. 2, PanelB). Of the three L^(d) binding peptides, in addition to IgE366, IgE11also induces antigen specific CTLs. The killing of IgE specific CTL isporeforin dependent and is independent of the expression of IFNγ (FIG.2, Panel C). Moreover, the CTL induced by IgE peptides are MHCrestricted because the killing of IgE44 loaded RMAS targets by IgE44specific CTL was completely blocked by anti-D^(b) mAb (FIG. 2, Panel D).FACS analysis of these CTL revealed that they are αβ TCR positive CD8⁺ Tcells and no expression of NK cell marker (DX5 or NK1.1) were detectedon these cells (data not shown).

Inhibition of IgE Responses by Anti-IgE Specific CTLs.

Because CTLs induced by IgE peptides kills the target cell specificallyin vitro, we were interested in seeing if these CTLs could inhibit theIgE responses in vivo. Mice have very low serum IgE and do not developallergic response spontaneously. Ovalbumin precipitated with AlumHydroxyde has been used to induce antigen specific IgE responses inmice. As shown in FIG. 3, after two immunizations with OVA plus alumhydroxyde, both total serum IgE and ova-specific IgE in the immunizedmice were high and the IgE level was further increased after intranasalchallenge of these mice with OVA.

TABLE 2 The Effect of Anti-IgE CTL on Airway Inflammation^(a)Eosinophilic Hyperplasia Treatment Inflammation^(b) infiltration ofBALT^(c) PBS (5) 3, 1, 2, 2, 0 2, 0, 2, 3, 0 2, 0, 2, 3, 0 Anti-IgECTL(5) 0, 0, 0, 1, 0 0, 0, 0, 0, 0 0, T, 0, 1, 0 Control CTL(4) 3, 3, 2,3 2, 3, 3, 1 3, 2, 2, 2 Normal mice(4) 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0^(a)Adult CBF1/J mice were immunized with 50 μg ovalbumin (OVA) plusAlum hydroxide intraperitoneally on Day 1 and Day 14. Two weeks afterthe second immunization, 5 × 106 anti-IgE CTL or a control CTL or PBSwere given every other day for three times. Three weeks after the lastCTL treatment, the mice were challenged with OVA intranasally everyother day for three times. One day after the last challenge, bronchialalveolar lavage was collected and lung tissue was collected from eachmice and stained with HE staining. The lung inflammation of each mousewas independently evaluated by a pathologist. ^(b)Score: O = Normal; T =trace; 1 = mild; 2 = mild to moderated; 3 = moderate; 4 = severe^(c)BALT = Bronchial Associated Lymphoid Hyperplasia.

TABLE 3 HLA-A2 Peptide Motif Search for Human IgE Score (Estimate HalfTime of Subsequence Disassociation of HLA-2 Start Residue Containingthis Rank Position Listing Subsequence) 1 185 WLSDRTYTC 93.696 2 96WVDNKTFSV 64.948 3 71 LLTVSGAWA 46.451 4 365 QLPDARHST 30.553 5 3TQSPSVFPL 28.893 6 309 ALMRSTTKT 27.572 7 59 TLTLSGHYA 27.324 8 54TLPATTLTL 21.362 9 47 SLNGTTMTL 21.362 10 61 TLSGHYATI 15.649 11 52TMTLPATTL 15.428 12 178 LTLSQKHWL 10.264 13 66 YATISLLTV 10.220 14 154QVMDVDLST 9.892 15 17 NIPSNATSV 9.563 16 133 LLCLVSGYT 9.058 17 403FICRAVHEA 7.227 18 236 TTTCLVVDL 6.756 19 356 SVQWLHNEV 6.086 20 155VMDVDLSTA 5.612

TABLE 4 HLA-A2 Peptide Motif Search for Human IgE by Neuro-Network NetOutput C150 Start End Sequence 0.747555 5.71921 223 231 RPSPFDLFI0.695169 8.21283 349 357 NFMPEDISV 0.628452 13.021 358 366 QWLHNEVQL0.60628 15.1782 33 41 GYFPEPVMV 0.53619 24.6281 54 62 TLPATTLT 0.4598141.7417 108 116 DFTPPTVKI 0.406526 60.3147 229 237 LFIRKSPTI 0.38260271.153 96 104 WVDNKTFSV 0.373791 75.6184 148 156 TWLEDGQVM 0.3498589.2174 61 69 TLSGHYATI 0.348214 96.2317 396 404 EWEQKDEFI 0.34468392.4594 278 286 LTVTSTLPV 0.317372 111.656 128 136 PPTIQLLCL 0.29653128.947 170 178 ELASTQSEL 0.292132 132.924 236 244 TITCLVVDL 0.272911151.798 106 114 SRDFTPPTV 0.26747 157.612 213 221 NPRGVSAYL 0.252711174.529 10 18 PLTRCCKNI 0.227935 207.107 147 155 ITWLEDGQV 0.220931217.374 234 242 SPTITCLVV 0.219179 220.02 47 55 SLNGTTMTL 0.218951220.368 384 392 FFVFSRLEV 0.199355 252.309 139 147 GYTPGTINI 0.188573271.82 123 131 GGGHFPPTI 0.170795 307.296 245 253 APSKGTVNL 0.136633389.134 302 310 THPHLPRAL 0.124225 423.96 284 292 LPVGTRDWI 0.115665449.785 378 386 KTKGSGFFV

Example 4

In the presence of specific antigen and constimulation, resting CD8 Tcells can be activated and differentiated into CTL, which plays anessential role in anti-virus immune response. Recently, it has also beenshown that tumor associated antigens specific CTL generated in vitro canbe used in treating cancer patients. Here we show that antigenicpeptides identified from non-tumor self-antigens can induce specificcytotoxic T lymphocyte (CTL) in vitro. The CTL induced by peptidesidentified from CD40L, a self antigen transiently expressed on activatedCD4 T cells, can kill activated CD4 T cells and the killing can beblocked either by the antibody (Ab) specific for the restricting class Imolecule or by the Ab recognizing CD8 molecule. In addition, neitheractivated CD4 T cells generated from CD40L^(−/−) mice nor from 2 m^(−/−)mice are killed by the CD40L specific CTL, demonstrating that thekilling of activated CD4 T cells by CD40L specific CTL isantigen-dependent and MHC restricted. Importantly, in vitro generatedCTL specific for CD40L inhibit CD4-dependent antibody responses of allisotypes in vivo. In contrast, CTL induced by antigenic peptides derivedfrom IgE specifically inhibit IgE responses and adoptive transfer ofCD40L-specific CU to NOD mice at early age delay the development ofdiabetes in NOD mice. Thus, in vitro generated CTL specific fornon-tumor self-antigens expressed on activated CD4 T cells can regulateimmune responses in vivo.

Allergic diseases, such as hay fever, asthma and systemic anaphylaxis,are immune responses to innocuous substances. The hallmark of thediseases is activation of CD4 cells and over production of IgE by Bcells. The current therapies have been focused on the treatment ofsymptoms and do not prevent the development and progression of thediseases. Because allergen-activated CD4 cells and IgE producing B cellsplay a central role in the pathogenesis of allergy, our strategy is touse autologous CTL to eliminate activated CD4 T cells and IgE producingB cells, thus preventing the development and progression of thediseases. Two molecules, CD40 ligand (CD40L) and IgE, were selected astarget antigens for CU therapy. Three antigenic peptides from CD40L andtwo antigenic peptides from IgE were identified. CTLs specific for thesepeptides have been generated and the function of these CTLs has beenevaluated both in vitro and in vivo.

Three antigenic epitopes from CD40L and two epitopes from IgE moleculeswere identified. Synthetic peptides of the antigenic epitopes were ableto bind to class I molecules and to activate resting naive CD8 T cellsin vitro.

CTLs were generated by stimulation of CD8 T cells with CD40L or IgEpeptides presented by Drosophila cells expressing MHC class I, B7-1 andICAM-1 molecules. The CTLs thus generated in vitro killed peptide-loadedtarget cells specifically. CD40L-peptide specific CTL killed activatedCD4 T cells and the recognition was dependent on the expression of CD40Land MHC class I molecules.

The function of CD40L-specific CTL were also evaluated in vivo.Antigen-specific antibody response was inhibited by anti-CD40L CTL. Theeffect of anti-CD40L CTL and anti-IgE CTL on allergy and autoimmunediseases will be investigated in animal models.

TABLE 5 MHC Class I Binding Motif Search for Mouse CD40L Sequence StartAA Identification Score Rank Position Sequence Number Number 1 17LPASMKIFM SEQ ID NO: 15 150.00 (L^(d)) 2 186 RPFIVGLWL SEQ ID NO: 16150.00 (L ^(d)) 3 118 DPQIAAHVV SEQ ID NO: 17 90.00 (L^(d)) 4 220QSVHLGGVF SEQ ID NO: 18 50.00 (L^(d)) 5 9 SPRSVATGL SEQ ID NO: 19 45.00(L^(d)) 6 195 KPSIGSERI SEQ ID NO: 20 39.00 (L^(d)) 7 252 FSSFGLLKL SEQID NO: 21 32.50 (L^(d)) 8 7 QPSPRSVAT SEQ ID NO: 22 30.00 (L^(d)) 9 181EPSSQRPFI SEQ ID NO: 23 30.00 (L^(d)) 10 79 LSLLNCEEM SEQ ID NO: 2425.00 (L^(d)) 1 79 LSLLNCEEM SEQ ID NO: 24 5713.03 (D ^(b)) 2 152VMLENGKQL SEQ ID NO: 25 5160.15 (D^(b)) 3 146 TMKSNLVML SEQ ID NO: 262648.88 (D^(b)) 4 235 SVFVNVTEA SEQ ID NO: 27 95.12 (D ^(b)) 5 38GSVLFAVYL SEQ ID NO: 28 46.87 (D^(b)) 6 19 ASMKIFMYL SEQ ID NO: 29 46.87(D^(b)) # Estimate of half time of disassociation of a moleculecontaining this subsequence.

TABLE 6 HLA-A2 Peptide Motif Search for Human CD40L Score SubsequenceSequence (Estimate of Half-Time of Start Residue IdentificationDisassociation of a Molecule Rank Position Listing Number Containingthis Subsequence) 1 24 FMYLLTVFL SEQ ID NO: 30 1249.083 2 167 GLYYIYAQVSEQ ID NO: 31 333.850 3 22 KIFMYLLTV SEQ ID NO: 32 284.846 4 36MIGSALFAV SEQ ID NO: 33 216.879 5 58 NLHEDFVFM SEQ ID NO: 34 212.854 6170 YIYAQVTFC SEQ ID NO: 35 127.199 7 26 YLLTVFLIT SEQ ID NO: 36 98.8038 231 LQPGASVFV SEQ ID NO: 37 65.934 9 45 YLHRRLDKI SEQ ID NO: 38 54.08610 147 TMSNNLVTL SEQ ID NO: 39 35.485 11 229 FELQPGASV SEQ ID NO: 4023.018 12 160 QLTVKRQGL SEQ ID NO: 41 21.362 13 35 QMIGSALFA SEQ ID NO:42 19.734 14 185 SQAPFIASL SEQ ID NO: 43 18.930 15 19 ISMKIFMYL SEQ IDNO: 44 9.166 16 153 VTLENGKQL SEQ ID NO: 45 7.652 17 126 VISEASSKT SEQID NO: 46 7.142 18 227 GVFELQPGA SEQ ID NO: 47 6.594 19 20 SMKIFMYLL SEQID NO: 48 4.720 20 165 RQGLYYIYA SEQ ID NO: 49 4.156

TABLE 7 Summary of CTL Activity Generated From PBMC in Different DonorsSequence Identification Specific IgE Peptide AA Sequence NumberKilling * 47 SLNGTTMTL¹ SEQ ID NO: 50 7/8 96 WVDNKTFSV¹ SEQ ID NO: 513/8 185 WLSDRTYTC SEQ ID NO: 52 0/8 308 ALSDRTYTC SEQ ID NO: 53 0/3 876SLLTVSGAWA SEQ ID NO: 54 0/5 883 WLEDGQVMDV SEQ ID NO: 55 1/5 884TLTVTSTLPV² SEQ ID NO: 56 8/8 887 QMFTCRVAHT SEQ ID NO: 57 1/4 890YATISLLTV¹ SEQ ID NO: 58 4/5 895 TLACLIQNFM² SEQ ID NO: 59 3/4 898QVMDVDLSTA² SEQ ID NO: 60 3/4 x/N x: number of donor from whom anti-IgECTL was generated; N: number of donor tested CD8+ T cells were purifiedfrom PBMC and cultured with Drosophila cells transfected with A2.1, B7.1and ICAM-1 in the presence of IgE peptides. Statistics indicated thecapability of IgE peptide to generate specific CTL response fromdifferent donors. ¹ and ² indicate anti-IgE CTL was generated from 9-merand 10-mer respectively.

1. A method for producing CTL specific for one or more non-tumor selfantigen T cell epitopes, comprising: a) isolating CD8+ T cells from asubject; b) loading antigen presenting cells (APC's) having Class I MHCmolecules with the T cell epitopes; c) culturing the CD8+ T cells withthe APC's for a period of time sufficient for activation of precursorCD8+ T cells specific for the T cell epitopes; d) expanding in culturethe activated CD8+ T cells in the presence of components required forproliferation of the activated CD8+ T cells; and e) collecting CD8+ Tcells from the culture.
 2. The method of claim 1, wherein the T cellepitopes are present on IgE protein.
 3. The method of claim 1, whereinthe T cell epitopes are present on CD40 ligand protein.
 4. CD8+ T cellsthat are specifically cytotoxic for a disease causing target cell,wherein the target cell has on its surface one or more non-tumor selfantigen T cell epitopes associated with Class I MHC molecules, andwherein the CD8+ T cells have been selectively activated by interactionwith Class I MHC molecules associated with the non-tumor self antigen Tcell epitopes.
 5. The method of claim 4, wherein the T cell epitopes arepresent on IgE protein.
 6. The method of claim 4, wherein the T cellepitopes are present on CD40 ligand protein.
 7. A method for treating adisease mediated by a disease causing target cell, wherein the targetcell has on its surface one or more non-tumor self antigen T cellepitopes associated with Class I MHC molecules, comprising administeringto a patient in need of such treatment, activated CD8+ T cells whereinthe CD8+ T cells have been selectively activated by interaction withClass I MHC molecules associated with the non-tumor self antigen T cellepitopes.
 8. The method of claim 7, wherein the T cell epitopes arepresent on IgE protein.
 9. The method of claim 7, wherein the T cellepitopes are present on CD40 ligand protein.